相关实验视频

Updated: Jun 19, 2025

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

在I型CRISPR-Cas系统中以HNH为媒介的目标DNA裂变的机制

Chendi Zhang¹, Fugen Chen¹, Feng Wang¹

¹State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan 430062, Hubei, China.

|July 24, 2024

概括

新发现的CRISPR相关的抗病毒防御 (级联) 综合体精确地切割DNA. 结构研究揭示了目标DNA结合和crRNA定位如何使HNH域解锁用于基因组编辑.

科学领域:

分子生物学分子生物学
遗传学是一个遗传学.
生物化学生化学

背景情况:

最近发现的与CRISPR相关的 (级联) 复合体是基因组编辑工具.
类型I-E和类型I-F级联变体与HNH域融合,精确地切割目标DNA.
这些基因组编辑工具的工作机制在很大程度上是未知的.

研究的目的:

阐明I-F型HNH和I-E型HNH级联综合体的工作机制.
确定这些复合体在不同功能状态下的结构.
为了确定控制其特定地点DNA裂变的关键因素.

主要方法:

使用X射线晶体学来确定I-F型HNH和I-E型HNH级联综合体的结构.
对不同状态的目标DNA相互作用的复合体进行了结构分析.
对两种复杂类型进行了比较结构分析.

主要成果:

结构揭示了Cas8f HNH在I-F类型的布中松散地附着在头部,并在R循环形成后重新定位,用于断目标DNA.
结构显示Cas5e HNH在I-E类型的布中通过布头的结构变化促进了目标DNA的结合.
这两种复合体都利用crRNA引导的向DNA定位和向DNA诱导的HNH解锁进行裂变.

结论:

目标DNA的crRNA引导定位对于特定部位的裂变至关重要.
目标DNA诱导的HNH解锁是这些CRISPR系统精确地分离DNA的关键机制.
这些发现提供了对新型基因组编辑工具的机制性见解.

关键词:

这就是CRISPR-Cas.HNH HNH HNH 的意思是什么?这是一个R环.一个布式的布式.基因组编辑基因组编辑特定地点的切割裂变.

更多相关视频

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Published on: August 25, 2017

相关概念视频

CRISPR and crRNAs

CRISPR and crRNAs

Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...

CRISPR

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...

Homologous Recombination

Homologous Recombination

The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...

Conservative Site-specific Recombination and Phase Variation

Conservative Site-specific Recombination and Phase Variation

Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...

Caspases

Caspases

Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. This gene encodes the ced-3 caspase that is similar to the interleukin-1-beta converting enzyme or ICE in mammals. In addition to apoptosis, caspases also function in the inflammatory response. Inflammatory caspases are essential in activating pro-inflammatory cytokines that recruit immune cells and block the replication of pathogens inside...

DNA Helicases

DNA Helicases

DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...